[Establishment of animal model for elucidating the mechanism of intoxication by the poisonous mushroom Clitocybe acromelalga].

Dietary intake of a poisonous mushroom, Clitocybe acromelalga, causes acromelalgia. The symptom continues for over a month. Some papers reported that treatment with nicotinic acid is effective. We have established an animal model to elucidate the mechanism of toxicity of the poisonous mushroom Clitocybe acromelalga. Diet containing Clitocybe acromelalga was fed to niacin-deficient rats for 24 hours (designated as day 0). The food intake decreased to about one-half compared with that of day before, and body weight loss was noted. Although the diet was returned to the control diet on day 1, the food intake did not recover until day 7, and body weight gain was not seen until day 6. A severe symptom resembling acromelalgia in humans started to appear on day 3. This is the first report of an animal model for the intoxication of Clitocybe acromelalga in humans. Since no similar symptom resembling human intoxication was seen in a previous rodent study, the niacin-free/tryptophan-limited diet used in the present study may have contributed to the result.

[Effect of feeding with a poisonous mushroom Clitocybe acromelalga on the metabolism of tryptophan-niacin in rats].

The poisonous mushroom Clitocybe acromelalga contains clitidine, which resembles nicotinic acid mononucleotide, and 4-amino-pyridine-2,3-dicarboxylic acid, which resembles quinolinic acid. Both are important intermediates in the tryptophan-niacin pathway. Therefore, we investigated the effect of feeding a niacin-free and tryptophan-limited diet containing the toadstool Clitocybe acromelalga on the metabolism of tryptophan to niacin in rats. The toadstool diet was fed to the rats for only one day (this day was designated day 0). Urinary excretion of intermediates in the tryptophan-niacin pathway, such as anthranilic acid, kynurenic acid, xanthurenic acid, 3-hydroxyanthranilic acid, quinolinic acid, nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide, was higher in the toadstool group than in the control on day 0-day 1 and day 1-day 2. The blood levels of tryptophan and NAD on day 1 were also higher in the toadstool group. Accordingly, intake of Clitocybe acromelalga appeared to increase the conversion of tryptophan to niacin.

Stimulatory effect of menaquinone-7 (vitamin K2) on osteoblastic bone formation in vitro.

Menaquinone-7, which is vitamin K2 (menatetrenone) with seven isoprene units, is highly contained in the fermented soybean. The effect of menaquinone-7 (MK-7) on osteoblastic bone formation was investigated. Femoral-diaphyseal and metaphyseal tissues of young male rats (4 weeks old) were cultured for 48 h in a medium containing either vehicle or MK-7 (10(-7)-10(-5) M). Calcium content, alkaline phosphatase activity, and deoxyribonuclic acid (DNA) content in the diaphyseal and metaphyseal tissues was significantly increased in the presence of MK-7 (10(-6) and 10(-5) M). The effect of MK-7 in increasing the diaphyseal and metaphyseal calcium content and alkaline phosphatase activity was completely prevented in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis. Moreover, osteoblastic MC3T3-E1 cells after subculture were cultured for 24 h in a serum-free medium containing MK-7 (10(-7)-10(-5) M). Protein content, alkaline phophatase activity, osteocalcin and DNA content in the cells was significantly increased in the presence of MK-7 (10(-6) and 10(-5) M). The effect of MK-7 in increasing protein content, alkaline phosphatase activity, and osteocalcin production in the cells was completely blocked by cycloheximide. This study demonstrates that MK-7 has an anabolic effect on bone tissue and osteoblastic MC3T3-E1 cells in vitro, suggesting that the compound can stimulate osteoblastic bone formation.

Increased conversion ratio of tryptophan to niacin by dietary di-n-butylphthalate.

We reported that the growth promoting activity of di-n-butylphthalate (DBP) was observed when rats were fed with a niacin-free and tryptophan-limiting diet (Shibata et al.. 1982. J Nutr Sci Vitaminol 28, 173-177). The present experiment was performed to investigate whether this phenomenon is attributable to the increase in the conversion ratio of tryptophan to niacin. The weaning rats were fed with a 10% (low protein diet) or 20% protein (conventional protein diet) diet with or without adding 1% DBP. The conversion ratio of tryptophan to niacin was significantly higher in the DBP group than in the control group; for 10% casein diets, it increased two-fold and for 20% casein diet, about five-fold. From these results, the previous finding is possibly explained by DBP increasing the conversion ratio of tryptophan to niacin.

Elevation of blood NAD level after moderate exercise in young women and mice.

We previously reported that the blood NAD levels are decreased by severe exercise, and administration of nicotinamide, a precursor of NAD, improves the endurance capacity of mice. In the present study, we determined whether moderate exercise changes the blood NAD levels in humans and mice. College female students exercised moderately with bike-ergometers. The blood NAD levels elevated after moderate exercise. Mice were forced to swim in a running water pool for 5 min as a moderate exercise, 15 min as a strong exercise, and until exhaustion as a severe exercise (average swimming time was 28.7 min). A 5 min swim gave a result similar to that of moderate exercise by human subjects. However, the blood NAD levels decreased after all-out exercise. The changes in whole blood tryptophan (a precursor of pyridine nucleotides) levels were similar to that in NAD. The glucose levels in whole blood and the non-esterified fatty acid levels in serum decreased according to exercising time. These data are the first demonstration of moderate exercise raising the blood NAD levels in human and mice. Elevation of the blood NAD levels may reflect changes in niacin metabolism that occur in response to exercise.

Effects of dietary pyrazinamide, an antituberculosis agent, on the metabolism of tryptophan to niacin and of tryptophan to serotonin in rats.

The effects of pyrazinamide on the metabolism of tryptophan to niacin and of tryptophan to serotonin were investigated to elucidate the mechanism for pyrazinamide action against tuberculosis. Weanling rats were fed with a diet with or without 0.25% pyrazinamide for 61 days. Urine samples were periodically collected for measuring the tryptophan metabolites. The administration of pyrazinamide significantly increased the metabolites, 3-hydroxyanthranilic acid and beyond, especially quinolinic acid, nicotinamide, N'-methylnicotinamide, and N1-methyl-4-pyridone-3-carboxamide, and therefore significantly increased the conversion ratio of tryptophan to niacin and the blood NAD level . However, no difference in the upper metabolites of the tryptophan to niacin pathway such as anthranilic acid, kynurenic acid and xanthurenic acid was apparent between the two groups. No difference in the concentrations of trytptophan and serotonin in the blood were apparent either. It is suggested from these results that the action of pyrazinamide against tuberculosis is linked to the increase in turnover of NAD and to the increased content of NAD in the host cells.

Effect of dietary histidine content on the change in content of skin urocanic acid isomers in hairless mice irradiated with ultraviolet B.

Hairless mice were fed with a 10% amino acid mixture diet (control diet, 0.42% histidine content), the control diet without histidine (histidine-free diet), or the control diet rich in histidine (histidine-rich diet; histidine content, 4.2%) for 32 days. They were irradiated with UV light of 312 nm for 30 min, and skin samples were periodically taken for measuring the urocanic acid isomers. Total urocanic acid isomers were decreased by UV irradiation in all the three groups, the recovery being the fastest in the histidine-rich group. The percentage increase in cis-urocanic acid/total urocanic acid was quickly increased by UVB irradiation. The recovery of the ratio was slightly higher in the histidine-rich group, although the total urocanic acid level was higher in the histidine-rich group than in the others. Therefore, the absolute cis-urocanic acid content in the skin was almost the same among the three groups. These results indicate that the increased histidine intake strengthened UVB protection without any decrease in immune suppression.

Changes of pyridine nucleotide levels during adipocyte differentiation of mouse 3T3-L1 cells.

The levels of NAD and NADP were measured in 3T3-L1 cells during a differentiation from preadipocytes to adipocytes. The cells were grown in the ordinary medium and differentiated in the medium by adding dexamethasone, 1-methyl-3-isobutylxanthine, and insulin for 2 days, and then they were grown in the medium by adding only insulin for another 8 days to accumulate fat. The levels of cellular NAD and NADP increased abruptly with days after differentiation, and the levels of NAD and NADP reached maximum at day 7, and at day 10 the values were decreased compared with the maximum values. These results suggest that expression of the pyridine nucleotide biosynthesis genes is induced in the differentiation process.

Influence of adenine-induced renal failure on tryptophan-niacin metabolism in rats.

To discover the role of the kidney in tryptophan degradation, especially tryptophan to niacin, rat kidneys were injured by feeding a diet containing a large amount of adenine. The kidney contains very high activity of aminocarboxymuconate-semialdehyde decarboxylase (ACMSD), which leads tryptophan into the glutaric acid pathway and then the TCA cycle, but not to the niacin pathway. On the other hand, kidneys contain significant activity of quinolinate phosphoribosyltransferase (QPRT), which leads tryptophan into the niacin pathway. The ACMSD activity in kidneys were significantly lower in the adenine group than in the control group, while the QPRT activity was almost the same, however, the formations of niacin and its compounds such as N1-methylnicotinamide and its pyridones did not increase, and therefore, the conversion ratio of tryptophan to niacin was lower in the adenine group than in the control group. The contents of NAD and NADP in liver, kidney, and blood were also lower in the adenine group. The decreased levels of niacin and the related compounds were consistent with the changes in the enzyme activities involved in the tryptophan-niacin metabolism in liver. It was concluded from these results that the conversion of tryptophan to niacin is due to only the liver enzymes and that the role of the kidney would be extremely low.

Efficiency of D-tryptophan as Niacin in rats.

L-tryptophan is a very important precursor of niacin in mammals. Food preparation in which proteins are exposed to an alkali and/or high temperature for a long period generate appreciable amounts of D-amino acids from racemization. The efficiency of D-tryptophan as niacin was thus investigated by using weanling rats. The availability of D-tryptophan was almost the same as that in L-tryptophan as the precursor of niacin and was 1/6 as active as niacin.

Anabolic effect of genistein in osteoblastic MC3T3-E1 cells.

Genistein is a natural isoflavone found in Leguminosae. The effect of genistein on osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 48 h in the presence of genistein (10(-7)-10(-5) M). Genistein (10(-6) and 10(-5) M) caused a significant elevation of protein content, alkaline phosphatase activity, and deoxyriboncleic acid (DNA) content in the cells. The effect of genistein (10-5 M) in increasing protein content, alkaline phosphatase activity and DNA content in the cells was completely prevented by the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis, suggesting that the isoflavone's effect results from a newly synthesized protein component. The effect of genistein (10(-5) M) in elevating cellular protein content and alkaline phosphatase activity was completely inhibited by the presence of trifluo-perazine (10(-5) M), staurosporine (10(-7) M) or vanadate (10(-6) M), various protein kinase inhibitors. Moreover, genistein (10(-5) M)-increased protein content and alkaline phosphatase activity in the cells was clearly abolished by the presence of anti-estrogen tamoxifen (10(-6) M). The effect of 17beta-estradiol (10(-9) M) in elevating protein and alkaline phosphatase activity in the cells was not enhanced by the presence of genistein (10(-5) M). Genistein's effect might be partly involved in estrogen action. The present study demonstrates that genistein has an anabolic effect on osteoblastic MC3T3-E1 cells.

Stimulatory effect of Daidzein in osteoblastic MC3T3-E1 cells.

Daidzein is a natural isoflavone found in Leguminosae. The effect of daidzein on osteoblastic MC3T3-E1 cells was investigated. Cells were cultured in a serum-free medium for 48 hr in the presence of daidzein (10(-7)-10(-5) M). Daidzein (10(-6) and 10(-5) M) caused a significant elevation of protein content, alkaline phosphatase activity, and DNA content in cells; those increases were about 1.4-, 1.5-, and 2.0-fold, respectively. The ability of daidzein (10(-5) M) to increase protein content, alkaline phosphatase activity, and DNA content in cells was prevented completely by the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis, indicating that the isoflavone has a stimulatory effect on cellular protein synthesis. The anabolic effect of daidzein (10(-6) or 10(-5) M) in cells was not distinguishable from that of genistein (10(-6) or 10(-5) M). Meanwhile, cell protein showed an additive effect of 17beta-estradiol and daidzein, but their effects on alkaline phosphatase were not additive. Moreover, the effect of daidzein (10(-5) M) in elevating cellular protein content and alkaline phosphatase activity was inhibited completely by the presence of tamoxifen (10(-6) M), suggesting that the effect of the isoflavone is mediated partly through estrogen action. This study demonstrates that daidzein has an anabolic effect in osteoblastic MC3T3-E1 cells.

Reversed-phase high-performance liquid chromatography of nicotinic acid mononucleotide for measurement of quinolinate phosphoribosyltransferase.

A system has been developed for the determination of quinolinate phosphoribosyltransferase (QPRT) activity in liver and kidney homogenates using HPLC. A product, nicotinic acid mononucleotide (NaMN), is separated by reversed-phase chromatography (a Tosoh ODS 80TS was used as an analytical column) using a mixture of 10 mM KH2PO4-K2HPO4 buffer (pH 7.0) containing 1.48 g/l tetra-n-butylammonium bromide-acetonitrile (9:1, v/v) as a mobile phase. The flow-rate was 1.0 ml/min, the detection wavelength was 265 nm. The column temperature was maintained at 40 degrees C. Under these conditions, NaMN was eluted at about 8.1 min. Sample preparation was very straightforward. The reaction mixture of QPRT assay was stopped by immersing the tube into a boiling water bath, the resulting supernatant was filtered, and the filtrate was directly injected into a HPLC system. The total HPLC analysis time was approximately 20 min.

Stimulatory effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells: activation of aminoacyl-tRNA synthetase.

The effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells in vitro was investigated to determine a cellular mechanism by which the isoflavones stimulate bone formation. Cells were cultured for 48 h in alpha-minimal essential medium containing either vehicle, genistein (l0(-7) - 10(-5) M) or daidzein (10(-7) - 10(-5) M). The 5,500 g supernatant of cell homogenate was used for assay of protein synthesis with [3H]leucine incorporation in vitro. The culture with genistein or daidzein caused a significant elevation of protein synthesis in the cell homogenate. The effect of genistein ( 10(-5) M) or daidzein ( 10(-5) M) in elevating protein synthesis was significantly prevented, when cells were cultured for 48 h in a medium containing either actinomycin D (10(-7) M) or cycloheximide (10(-6) M) in the absence or presence of isoflavones. Moreover, when genistein (10(-7) 10(-5) M) or daidzein (10(-6) and 10(-5) M) was added to the reaction mixture containing the cell homogenate obtained from osteoblastic cells cultured without isoflavone, protein synthesis was significantly raised. This increase was markedly blocked by the addition of cycloheximide (10(-7) M). In addition, [3H]leucyl-tRNA synthetase activity in the cytosol of osteoblastic cells was significantly increased by the addition of genistein (10(-6) and 10(-5) M) or daidzein (10(-5) M) into the enzyme reaction mixture. The present study demonstrates that genistein or daidzein can stimulate protein synthesis in osteoblastic MC3T3-E1 cells. The isoflavones may have a stimulatory effect on osteoblastic bone formation due to increasing protein synthesis.

Carotenoids and retinoids as suppressors on adipocyte differentiation via nuclear receptors.

The adipocyte differentiation program is regulated by the sequential expression of transcriptional activators, mainly peroxisome proliferator activated receptor (PPAR) families. In the present study, we have decided to systematically examine the effects of vitamin A and its precursors, carotenoids and retinoids, on terminal differentiation from preadipocytes to adipocytes on the cellular and molecular aspects. The effects of active form of vitamin A, retinoic acid (RA), are believed to be mediated by specific nuclear receptor proteins [retinoic acid receptor (RAR)] which are members of the steroid and thyroid/retinoid receptor superfamily of ligand dependent transcriptional regulators, RARalpha, RARgamma, RXRalpha, and RXRbeta mRNA were abundant in adipose tissue and 3T3-L1 adipose cells. The autoregulated amplification of RARgamma mRNA was observed by these own ligands in 3T3-L1 cells. And, RA inhibited PPARgamma2 expression more effectively and caused concomitantly a greater inhibition of adipocyte differentiation. These results suggest that the inhibitory action of adipocyte differentiation by carotenoids and retinoids are exhibited through the RAR up-regulation and the suppression of PPARgamma2. The nature of the cross talk of vitamin A actions between the RARs, RXRs and PPARs via co-activator in adipose tissue will likely prove to be important for understanding the process of adipogenesis.

The inhibitory effect of recombinant human soluble thrombomodulin on initiation and extension of coagulation--a comparison with other anticoagulants.

Recombinant human soluble thrombomodulin (rhsTM) was compared with various anticoagulants for in vitro anticoagulant effects on thrombin generation, clotting time, and thromboelastography. rhsTM as well as APC reduced the level of the peak of the thrombin generation curve, but we did not observe any time-delay to reach the peak. This effect of rhsTM was diminished in PC-deficient plasma and was closely associated with the inhibitory effect on prothrombinase and factor Va. On the other hand, hirudin and argatroban delayed the time to reach the level of the peak, without reducing it. rhsTM and other anticoagulants except for activated protein C (APC) were found to have concentration-dependent anticoagulant activity by conventional clotting tests. However, the concentration of rhsTM for clotting time was slightly affected by anti-protein C antibody. Moreover, the concentration of rhsTM required to inhibit thrombin activity directly was 50 times higher than that needed to inhibit thrombin generation. The effect of rhsTM on clot development was compared with that of other anticoagulants by thromboelastography; rhsTM reduced the growth of the clot but had little effect on the time to activate clotting, while the other anticoagulants had the opposite effect. This effect of rhsTM was completely abolished by the addition of anti-protein C or anti-protein S antibody. These findings suggest that rhsTM attenuates blood clotting by reducing the level of generated thrombin through protein C activation and subsequent factor Va inactivation and prothrombinase inhibition.

A shear-induced in vitro platelet function test can assess clinically relevant anti-thrombotic effects.

Morphological features of haemostatic plugs formed in vitro under high shear forces were investigated. Electron microscopy confirmed the relevance of such haemostatic plug to a platelet-rich arterial thrombus, which is formed in vivo . In rat blood samples, the effects of anticoagulants and various antiplatelet agents on platelet reactivity (rate of haemostatic plug formation) and subsequent coagulation of the flowing blood were investigated. Haemostasis did not occur in citrated blood, and heparin greatly inhibited the shear-induced platelet reaction. Aspirin (1 mM), a thromboxane A(2) receptor antagonist (5 microM), a stable prostacyclin (0.55 nM), a stable prostaglandin E(1) (141 nM) and a phosphodiesterase inhibitor (100 microM) were tested. All these agents exerted significant inhibitory effect on shear-induced platelet reaction, including the inhibition of the very first phase of platelet plug formation, due to aggregation of shear-activated platelets. Except for the phosphodiesterase inhibitor, which prolonged clotting time, none of the above agents affected dynamic coagulation. These results suggest that the employed in vitro shear-induced thrombosis/haemostasis test can reveal in vivo the antithrombotic effect of various agents independently of their mechanism of action.

Effects of recombinant human soluble thrombomodulin (rhs-TM) on clot-induced coagulation in human plasma.

Recent studies have suggested that clot-bound thrombin plays an important role in thrombus growth. In this study, we examined the effects of recombinant human soluble thrombomodulin (rhsTM) on clot-induced coagulation. rhsTM enhanced the activation of protein C by clots, and attenuated clot-induced thrombin generation and fibrinopeptide A (FPA) production in a dose-dependent manner. The inhibitory effect of rhsTM was abolished by anti-protein C antibody. The inhibitory effect of rhsTM on clot-induced thrombin generation continued for over 60 min after the addition of the clot, while an active site-directed thrombin inhibitor, argatroban, produced a more transient inhibition. rhsTM also inhibited the regrowth of the clot in (125)I-fibrinogen-supplemented plasma. We also examined the effect of rhsTM by thromboelastography, rhsTM reduced the growth of the clot but had little effect on the time to begin clotting, while heparin and Fragmin (low molecular weight heparin) had effects opposite to those of rhsTM. These findings suggest that rhs-TM attenuates the growth of the clot by activating protein C and inhibiting further thrombin generation in the clot.

A case of extrahepatic portal vein aneurysm with massive thrombosis: diagnosis with reconstruction images from helical CT scans.

A patient with extrahepatic portal vein aneurysm with massive thrombosis, congenital in nature, is reported. Reconstructed images using curved reformation software and three-dimensional images from helical CT were useful to delineate the shape of the portal vein aneurysm and extension of the thrombi.